1. Field of Endeavor
The present invention relates to thermal cycling and more particularly to a rapid microfluidic thermal cycler.
2. State of Technology
U.S. Pat. No. 7,133,726 for a thermal cycler for PCR states: “Generally, in the case of PCR, it is desirable to change the sample temperature between the required temperatures in the cycle as quickly as possible for several reasons. First the chemical reaction has an optimum temperature for each of its stages and as such less time spent at non-optimum temperatures means a better chemical result is achieved. Secondly a minimum time is usually required at any given set point which sets minimum cycle time for each protocol and any time spent in transition between set points adds to this minimum time. Since the number of cycles is usually quite large, this transition time can significantly add to the total time needed to complete the amplification.” U.S. Pat. No. 7,133,726 includes the additional state of technology information below:                “To amplify DNA (Deoxyribose Nucleic Acid) using the PCR process, it is necessary to cycle a specially constituted liquid reaction mixture through several different temperature incubation periods. The reaction mixture is comprised of various components including the DNA to be amplified and at least two primers sufficiently complementary to the sample DNA to be able to create extension products of the DNA being amplified. A key to PCR is the concept of thermal cycling: alternating steps of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of double-stranded DNA. In thermal cycling the PCR reaction mixture is repeatedly cycled from high temperatures of around 90° C. for melting the DNA, to lower temperatures of approximately 40° C. to 70° C. for primer annealing and extension. Generally, it is desirable to change the sample temperature to the next temperature in the cycle as rapidly as possible. The chemical reaction has an optimum temperature for each of its stages. Thus, less time spent at non optimum temperature means a better chemical result is achieved. Also a minimum time for holding the reaction mixture at each incubation temperature is required after each said incubation temperature is reached. These minimum incubation times establish the minimum time it takes to complete a cycle. Any time in transition between sample incubation temperatures is time added to this minimum cycle time. Since the number of cycles is fairly large, this additional time unnecessarily heightens the total time needed to complete the amplification.        In some previous automated PCR instruments, sample tubes are inserted into sample wells on a metal block. To perform the PCR process, the temperature of the metal block is cycled according to prescribed temperatures and times specified by the user in a PCR protocol file. The cycling is controlled by a computer and associated electronics. As the metal block changes temperature, the samples in the various tubes experience similar changes in temperature. However, in these previous instruments differences in sample temperature are generated by non-uniformity of temperature from place to place within the sample metal block. Temperature gradients exist within the material of the block, causing some samples to have different temperatures than others at particular times in the cycle. Further, there are delays in transferring heat from the sample block to the sample, and those delays differ across the sample block. These differences in temperature and delays in heat transfer cause the yield of the PCR process to differ from sample vial to sample vial. To perform the PCR process successfully and efficiently and to enable so-called quantitative PCR, these time delays and temperature errors must be minimized to the greatest extent possible. The problems of minimizing non-uniformity in temperature at various points on the sample block, and time required for and delays in heat transfer to and from the sample become particularly acute when the size of the region containing samples becomes large as in the standard 8 by 12 microtiter plate.        Another problem with current automated PCR instruments is accurately predicting the actual temperature of the reaction mixture during temperature cycling. Because the chemical reaction or the mixture has an optimum temperature for each of its stages, achieving that actual temperature is critical for good analytical results. Actual measurement of the temperature of the mixture in each vial is impractical because of the small volume of each vial and the large number of vials.”        
United States Published Patent No. 2005/0252773 for a thermal reaction device and method for using the same includes the following state of technology information:                “Devices with the ability to conduct nucleic acid amplifications would have diverse utilities. For example, such devices could be used as an analytical tool to determine whether a particular target nucleic acid of interest is present or absent in a sample. Thus, the devices could be utilized to test for the presence of particular pathogens (e.g., viruses, bacteria or fungi), and for identification purposes (e.g., paternity and forensic applications). Such devices could also be utilized to detect or characterize specific nucleic acids previously correlated with particular diseases or genetic disorders. When used as analytical tools, the devices could also be utilized to conduct genotyping analyses and gene expression analyses (e.g., differential gene expression studies). Alternatively, the devices can be used in a preparative fashion to amplify sufficient nucleic acid for further analysis such as sequencing of amplified product, cell-typing, DNA fingerprinting and the like. Amplified products can also be used in various genetic engineering applications, such as insertion into a vector that can then be used to transform cells for the production of a desired protein product.”        
United States Published Patent No. 2008/0166793 by Neil Reginald Beer for sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture provides the following state of technology information:                “A complex environmental or clinical sample 201 is prepared using known physical (ultracentrifugation, filtering, diffusion separation, electrophoresis, cytometry etc.), chemical (pH), and biological (selective enzymatic degradation) techniques to extract and separate target nucleic acids or intact individual particles 205 (e.g., virus particles) from background (i.e., intra- and extra-cellular RNA/DNA from host cells, pollen, dust, etc.). This sample, containing relatively purified nucleic acid or particles containing nucleic acids (e.g., viruses), can be split into multiple parallel channels and mixed with appropriate reagents required for reverse transcription and subsequent PCR (primers/probes/dNTPs/enzymes/buffer). Each of these mixes are then introduced into the system in such a way that statistically no more than a single RNA/DNA is present in any given microreactor. For example, a sample containing 106 target RNA/DNA would require millions of microreactors to ensure single RNA/DNA distribution.        An amplifier 207 provides Nucleic Acid Amplification. This may be accomplished by the Polymerase Chain Reaction (PCR) process, an exponential process whereby the amount of target DNA is doubled through each reaction cycle utilizing a polymerase enzyme, excess nucleic acid bases, primers, catalysts (MgCl2), etc. The reaction is powered by cycling the temperature from an annealing temperature whereby the primers bind to single-stranded DNA (ssDNA) through an extension temperature whereby the polymerase extends from the primer, adding nucleic acid bases until the complement strand is complete, to the melt temperature whereby the newly-created double-stranded DNA (dsDNA) is denatured into 2 separate strands. Returning the reaction mixture to the annealing temperature causes the primers to attach to the exposed strands, and the next cycle begins.        The heat addition and subtraction powering the PCR chemistry on the amplifier device 207 is described by the relation:Q=hA(Twall−T∞)        The amplifier 207 amplifies the organisms 206. The-nucleic acids 208 have been released from the organisms 206 and the nucleic acids 208 are amplified using the amplifier 207. For example, the amplifier 207 can be a thermocycler. The nucleic acids 208 can be amplified in-line before arraying them. As amplification occurs, detection of fluorescence-labeled TaqMan type probes occurs if desired. Following amplification, the system does not need decontamination due to the isolation of the chemical reactants.”        
U.S. Pat. No. 3,635,037 for a Peltier-effect heat pump provides the following state of technology information:                “The Peltier-effect has been used heretofore in heat pumps for the heating or cooling of areas and substances in which fluid-refrigeration cycles are disadvantageous. For example, for small lightweight refrigerators, compressors, evaporators and associated components of a vapor/liquid refrigerating cycle may be inconvenient and it has, therefore, been proposed to use the heat pump action of a Peltier pile. The Peltier effect may be described as a thermoelectric phenomenon whereby heat is generated or abstracted at the junction of dissimilar metals or other conductors upon application of an electric current. For the most part, a large number of junctions is required for a pronounced thermal effect and, consequently, the Peltier junctions form a pile or battery to which a source of electrical energy may be connected. The Peltier conductors and their junctions may lie in parallel or in series-parallel configurations and may have substantially any shape. For example, a Peltier battery or pile may be elongated or may form a planar or three-dimensional (cubic or cylindrical) array. When the Peltier effect is used in a heat pump, the Peltier battery or pile is associated with a heat sink or heat exchange jacket to which heat transfer is promoted, the heat exchanger being provided with ribs, channels or the like to facilitate heat transfer to or from the Peltier pile over a large surface area of high thermal conductivity. A jacket of aluminum or other metal of high thermal conductivity may serve for this purpose.”        
International Patent Application No. WO2008070198 by California Institute of Technology published Jun. 12, 2008 entitled “thermal cycling system” provides the following state of technology information:                “Invented in 1983 by Kary Mullis, PCR is recognized as one of the most important scientific developments of the twentieth century. PCR has revolutionized molecular biology through vastly extending the capability to identify and reproduce genetic materials such as DNA. Nowadays PCR is routinely practiced in medical and biological research laboratories for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing. The method has been automated through the use of thermal stable DNA polymerases and a machine commonly referred to as “thermal cycler.”        The conventional thermal cycler has several intrinsic limitations. Typically a conventional thermal cycler contains a metal heating block to carry out the thermal cycling of reaction samples. Because the instrument has a large thermal mass and the sample vessels have low heat conductivity, cycling the required levels of temperature is inefficient. The ramp time of the conventional thermal cycler is generally not rapid enough and inevitably results in undesired non-specific amplification of the target sequences. The suboptimal performance of a conventional thermal cycler is also due to the lack of thermal uniformity widely acknowledged in the art. Furthermore, the conventional real-time thermal cycler system carries optical detection components that are bulky and expensive. Mitsuhashi et al. (U.S. Pat. No. 6,533,255) discloses a liquid metal PCR thermal cycler.        There thus remains a considerable need for an alternative thermal cycler design. A desirable device would allow (a) rapid and uniform transfer of heat to effect a more specific amplification reaction of nucleic acids; and/or (b) real-time monitoring of the progress of the amplification reaction in real time. The present invention satisfies these needs and provides related advantages as well.        In one embodiment, a thermal cycler body (101; 151) comprises a fan (103; 153) and a removable heat block assembly, or swap block (105; 155) (FIG. 1). The swap block (105; 155) is inserted into and removed from the thermal cycler body (103; 153) by optionally sliding the swap heat block on sliding rails (113;163). After the swap block (105; 155) is inserted into the thermal cycler body (103; 153) the door of the thermal cycler (115;165) may be closed. The swap heat block (105; 155) comprises a liquid composition container (111; 161) and a heat sink (107;157) and optionally capped samples (109;159). In one embodiment the swap heat block (FIG. 2) comprises a receptacle with wells that seals the in the liquid composition so that the sample vessels do not contact the liquid (metal, metal alloy or metal slurry). In another embodiment the swap block (105; 155) comprises a receptacle barrier with wells (307;407) that is sealed to a liquid composition container housing (311;411), wherein the seal is liquid tight and may optionally comprise a gasket (309;409), (FIGS. 3 and 4). Further, the liquid composition container housing (311;411) is sealed to a base plate (313;413), which may be a metal plate (such as copper or aluminum), wherein the seal is liquid tight and may optionally comprise a gasket (312;412). The base plate (313;413) is in turn thermally coupled to a Peltier element (315;415), heats and cools the liquid composition and is in turn coupled to a heat sink (417). Optionally, a heat spreader (such as a copper, aluminum, or other metal or metal alloy that has high thermal conductivity) is sandwiched between the base plate (313;413) and the Peltier element (315;415). In some embodiments the swap block (105; 155) is held together by fasteners, such as screws (301;401). In one embodiment the swap block comprises a first piece, such as a receptacle with 48 wells (307;407), that is occupied by a second piece, such as a sample vessel, including but not limited to a sample plate (305;405), a single sample vessel or a strip of sample vessels, into which a third piece, such as a transparent cap plate (303;403), a single cap or strip of caps is inserted In one embodiment the a transparent cap plate (303;403), a single cap or strip of caps optionally comprises an extrusion, such as a light guide.”        